This invention is in the field of chemistry, biology and microbiology and relates to methods and compositions for detecting the presence of vancomycin-resistant Enterococci in a sample of a possibly contaminated material.
Enterococci are gram-positive bacteria that inhabit the gastrointestinal tract of healthy individuals. These bacteria have been identified as opportunistic pathogens for humans. Diseases caused by Enterococci include endocarditis, enterococcal bacteremia, urinary tract infections, neonatal infections, central nervous system infections (rare), intraabdominal and pelvic infections. Enterococci have emerged as one of the leading causes of nosocomial infections, responsible for 10% of all infections acquired in the hospital (Emori, T. G. and Gaynes, R. P. Clin. Microbiol. Rev. 6:428-42, 1993).
Recent alert about Enterococci is caused not only by their increasing role in nosocomial infections, but also by their resistance to vancomycin, an antibiotic that has been used for treating infection caused by gram positive cocci. Vancomycin resistant Enterococci, emerged as the nosocomial pathogen of the 1990s, have only been discovered in the late 1980s. From 1989 through 1993, the percentage of nosocomial infections reported by the United States Center for Disease Control""s National Nosocomial Infections Surveillance system that were caused by vancomycin-resistant Enterococci increased from 0.3% to 7.9% (CDC, MMWR Report 1995). Vancomycin resistant Enterococci have raised the public""s anxieties and prompt intense infection control measures in hospitals around the world because no known effective therapy exists for life-threatening vancomycin-resistant enterococcal infections.
Statistics based on the United States Center for Disease Control indicated that 10% of the enterococcal infections are caused by vancomycin-resistant Enterococci with an approximate 60% of mortality rate. The World Health Organization acknowledged that vancomycin-resistant Enterococci are one of the most serious threats to human health. Recommendations for preventing the spread of vancomycin resistance have been extensively discussed in Infectious Disease Alert vol. 14, 185, 189, 1995; 44 MMWR, RR-12, 1995; Edmond et al., Clin. Inf. Dis. 20:1126, 1995; 59 Federal Register 25758, 1994; and 16 Infection Control Hospital Epidemiology, 105, 1995.
Prompt detection and reporting of vancomycin-resistant Enterococci isolates are critical for preventing endemic spread of vancomycin-resistance Enterococci and allowing proper treatment once the right drug becomes available.
Currently, numerous vancomycin-containing selective media are used in the surveillance of vancomycin-resistant Enterococci. There is, however not a commercially available method which allows accurate, easy, and rapid detection of this important nosocomial pathogen. Recently, Landman et al., J. Clin. Microbiol. (1996) 34:751-752, described the use of five selective media or identifying fecal carriage of vancomycin-resistant Enterococci.
A common procedure for detecting vancomycin resistant Enterococci by all these available methods involves adding a suspect specimen into a sterile culture medium containing all the necessary elements for bacterial growth. The media may be a liquid medium or a solid agar medium. The sample may be natural or pretreated, as by transporting the sample in a preservative medium before adding it to the selective culture medium and the medium often contain vancomycin to selective for vancomycin-resistant Enterococci. Usually, these culture media are sterilized to prevent interference from contaminating microbes, and an incubation period of from 48 to 72 hours are required for detection of vancomycin-resistant Enterococci.
One major problem for using these types of selective media is that many bacteria are intriscally resistant to vancomycin. Examples include almost of the gram negative bacteria and some gram positive bacteria (Lactobacillus spp. Pediococcus spp., and Leuconostoc spp.). Once growth is observed in these culture media, the target microbes must be isolated and confirmed through selective isolation and one or more tests specific for a variety of physiological and biochemical characteristics. Often, a number of specific colonies must be sequentially tested. In some cases, the overgrown gram negative bacteria (such as the swarming Proteus spp.) on the culture plates prevent accurate identification of specific colonies for subsequent tests. Additionally, the isolated cultures must be confirmed through antibiotic susceptibility tests for vancomycin resistance.
These methods are labor intensive, time consuming, and require highly skilled medical technologists or microbiologists to perform the tests. The above described methods usually take at least 2-3 days to complete, and are suspectible to false positives and false negatives.
The use of chromogenic or fluorogenic enzyme substrates have been widely used in microbial diagnostic methods. For example, Edberg (U.S. Pat. No. 4,925,789) described using a nutrient indicator which not only serves as a nutrient indicator, but also changes color upon being metabolized. This patent, herein incorporated by reference, provides a medium containing a nutrient indicator which, when metabolized by target bacteria, releases a moiety which imparts a color or other detectable change to the medium. The procedure takes advantage of enzyme specificity unique to particular speciies of groups of bacteria. It describes using antibiotics to select for growth of the target microorganisms and provides a specific example of liquid based assay.
Kilian et al. Acta Path. Microbiol. Scand. Sec. B xcex47 271-276 (1979) and Demare et al., J. Food Science 50:1736 (1985) report use of agar-based media without antibiotics. Chen and Gu, U.S. Ser. No. 08/335,149, filed Nov. 4, 1994, incorporated by reference herein, described the use of a fluorogenic nutrient indicator, 4-methylumbelifery-xcex2-D-glucopyranoside, in a microbe-specific medium for detecting Enterococci. Each of the above described methods, however, is not suitable for detecting vancomycin-resistant Enterococci.
The above discussion is not an admission that any of the references discussed is prior art to this invention.
The present invention provides a method and media for specific detection of target microbes in a clinical sample. One of the problems in clinical sampling is that many bacteria are physiologically or biochemically similar, since these organisms reside in the same ecological system such as gastrointential tract of humans. Therefore, a simple, single enzyme reaction is often insufficient to specifically detect an organism in a medium. To achieve specific detection of target microbes, at least two enzymes should be used.
According to the present invention, a medium is provided in the method of performing a microbial diagnostic test, in which target microbes metabolize at least two nutrient indicators to yield detectable signals, and in which the presence of target microbes is indicated by the detectable characteristics yielded by two specific enzymatic reactions. The specific enzymes include xcex2-glucosidase and pyrrolidonyl arylamidase.
Preferrably, the two or more nutrient indicators yield distinctively different detectable signals so that the presence of both or more detectable signals is distinctively detectable from the presence of only one or some of the detectable signals. In such a case, the two or more nutrient indicators can be detected at the same or about the same time. For example, one nutrient indicator gives a color in the visual range while another nutrient indicator produces fluorescence under a ultraviolet lamp.
However, in designing aqueous assay systems using two chromogenic or two fluorogenic compounds as nutrient indicators, it s often difficult or even impossible to find two nutrient indicators with different colored products, or whose signals do not interfere with each other. Obviously, two indicators which yield the same color would be useless for detecting the presence of both. A less obvious problem exists when a strong red colored product masks a light yellow signal, or when a blue colored product quenches a fluorescent signal. To overcome these problems, this invention uses a second nutrient indicator that produces a colorless intermediate product which, upon reacting with a developing agent, generates a second detectable signal, e.g. on a filter paper. This approach prevents interference from the colored product of the first nutrient indicator in the medium. Thus, specific detection of vancomycin-resistant Enterococci in a sample is achieved in this invention by using sequential detection of metabolic hydrolysis of two nutrient indicators in the medium.
Thus, in a first aspect, this invention features a medium for detecting two or more bacterial enzymes. The medium contains a first nutrient indicator for a first bacterial enzyme. The first nutrient indicator provides a first detectable signal when cleaved by the first bacterial enzyme. The medium also contains a second nutrient indicator for a second bacterial enzyme. The second nutrient indicator provides an intermediate molecule when cleaved by said second bacterial enzyme. The intermediate molecule provides a second detectable signal upon reacting with a developing agent.
In preferred embodiments, the first bacterial enzyme is xcex2-glucosidase and the first nutrient indicator may be selected from the group of xcex2-glucosidase substrates consisting of resofuran-xcex2-D-glucopyranoside, o-nitrophenyl-xcex2-D-glucopyranoside, p-nitrophenyl-xcex2-D-glucopyranoside, 5-bromo-4-chloro-3-indoxyl-xcex2-D-glucopyranoside, 6-bromo-2-naphtyl-xcex2-D-glucopyranoside, Rose-xcex2-D-glucopyranoside, VQM-Glc(2-{2-[4-(xcex2-D-glucopyranosyloxy)-3-methoxyl]vinyl)-1-methyl-quinolinium iodide, VBZTM-Gluc(2-{2-[4-(xcex2-D-glucopyranosyloxy)3-methoxylphenyl]vinyl}-3-methylbenzothiazolium iodide, and 4-methylumbelliferyl-xcex2-D-glucopyranoside. This list is not meant to exclude xcex2-glucosidase substrates which have yet to be discovered but may later be identified and included in this list by those of ordinary skill in the art.
In other preferred embodiments, the second enzyme is pyrrolidonyl arylamidase and the intermediate molecule alters the color of said medium upon reacting with a color developing agent. The second nutrient indicator is pyroglutamic acid-xcex2-naphtylamide, and the preferred color developing agent is p-dimethylaminocinnamaldehyde.
By xe2x80x9cmediumxe2x80x9d is meant a solid, semi-solid, powder or liquid mixture which contains all, substantially all, or some of the nutrients necessary to support bacterial growth. Amino acids, minerals, vitamins, and other elements known to those skilled in the art to be necessary for bacterial growth are provided in the medium, which include, but are not limited to, those disclosed in U.S. application Ser. No. 08/335,149, filed on Nov. 4, 1994, incorporated by reference herein. In a preferred embodiment, the medium is liquid. In another preferred embodiment, the medium is agar. In yet another preferred embodiment, the medium is in powder form which upon rehydration allows the growth and detection of bacteria such as vancomycin-resistant Enterococci. The medium of this invention is free of visable target microbes; otherwise, it may be sterile or non-sterile.
For example, the following components are provided in the medium in approximately the amounts indicated. Those in the art will understand that not every component is required. Components may also be substituted with other components of similar properties. The amounts of components may also be varied.
Amino acids may be provided from a variety of sources. These can be provided from natural sources (e.g., extracts of organisms), as mixtures, or in purified form. The natural mixtures may contain varying amounts of such amino acids and vitamins. Not all amino acids must be provided, and the relative amount of each can vary. For general guidance, specific amounts of such amino acids and vitamins are indicated below. These amounts are for guidance only and are not limiting in this invention. Those in the art will recognize that many different combinations of amino acids and vitamins can be used in the medium of this invention. The lists provided below exemplify just one such example. Normally, only amino acids which cannot be synthesized endogenously by the microorganisms to be detected must be provided. However, other amino acids may be provided without departing from the medium of the invention.
The medium preferably includes at least the following amino acids in approximately the following amounts (per liter of medium): alanine (0.1 to 0.3 grams), arginine (0.1 to 0.3 grams), aspartic acid (0.4 to 0.7 grams), cystine (0.01 to 0.015 grams), glutamic acid (1.0 to 1.6 grams), glycine (0.12 to 0.17 grams), histidine (0.116 to 0.17 grams), isoleucine (0.25 to 0.37 grams), leucine (0.4 to 0.6 grams), lysine (0.37 to 0.56 grams), methionine (0.13 to 0.19 grams), phenylalanine (0.2 to 0.3 grams), proline (0. 4 to 0.6 grams), serine (0.18 to 0.26 grams), theronine (0.19 to 0.28 grams), tryptophan (0.05 to 0.07 grams), tyrosine (0.12 to 0.18 grams), and valine (0.29 to 0.44 grams).
Salts may be provided as a source of ions upon dissociation. Such salts may include (per liter of medium): potassium chloride (e.g., about 0.5 to 1.5 grams); copper sulfate (e.g., about 40 to 50 xcexcg); ammonium sulfate (eg., about 4.0 to 6.0 grams); potassium iodide (e.g., about 50.0 to 150.0 xcexcg); manganese sulfate (e.g., about 300.0 to 500.0 xcexcg); sodium molybdate (e.g., about 150.0 to 250.0 xcexcg); zinc sulfate (e.g. about 300.0 to 500.0 xcexcg); and sodium chloride (e.g. about 0.05 to 0.15 g).
Other inorganic moieties may be included to aid microbial growth. These include the following (to the extent not already provided in the above sources of various chemical entities and described in amounts per liter): Phosphorus (about 0.5 mg), Potassium (about 0.4 mg), Sodium (about 30 to 60 mg), and trace amounts of Calcium, Magnesium, Aluminum, Barium, Chloride, Cobalt, Copper, Iron, Lead, Manganese, Suffate, Sulfur, Tin and Zinc.
Vitamins required for growth and reproduction of the microorganism sought to be detected may also be provided. These can be provided in a pure form or as part of a more complex medium. Vitamins may be present in approximately the following amounts (per liter of medium): biotin (about 220 to 330 xcexcg), pantothenic acid (about 44 to 6 xcexcg), pyridoxine (about 9 to 14 mg), riboflavin (about 11 to 17 mg), folic acid (about 6 to 8 mg), thiamine (about 16 to 24 mg), niacin (about 15 to 23 mg), and trace amount (less than 10 xcexcg) of cyanocobalamin.
The medium may also contain an agent which induces enzyme activity. This agent may be an analog to the nutrient indicator. For example, isopropyl-xcex2-D-thiogalactoside (IPTG) induces xcex2-galactosidase activity. Ethyl-xcex2-D-thioglucoside induces xcex2-glucosidase activity. L-pyroglutamamide, L-pyroglutamic acid, and pyroglutamic acid penta-chloropenyl ester induce pyrrolidonyl arylamidase activity.
By xe2x80x9cbacterial enzymexe2x80x9d is meant an enzyme whose enzymatic activity such as the ability to hydrolyse a substrate or a plurality of substrates is characteristic of a bacterium or a plurality of bacteria. In this invention, the enzymatic activities of a bacterial enzyme or bacterial enzymes are used to detect the presence or measure the concentration of bacteria in a test sample. The bacterial enzymes include all those known to one skilled in the art, including, but not limited to, those listed in Enzymes, 3rd edition, edited by Malcolm Dixson, Edwin C. Webb, C. J. R. Thorne, and K. F. Tipton, 1979, Academic Press, U.S.A. Examples include, but are not limited to, alkaline phosphatase, acid phosphatase, esterase, lipase, N-acetyl-xcex2-D-galactosaminidase, N-acetyl-xcex2-D-glucosaminidase, Neuraminidase, L-arabinopyranosidase, xcex2-D-fucosidase, xcex1-L-fucosidase, xcex2-L-fucosidase, xcex1-D-galactosidase, xcex2-D-galactosidase, xcex1-D-glucosidase, xcex2-D-glucosidase, xcex2-D-glucuronidase, pyrrolidonyl arylamidase, xcex1-D-mannosidase, pyrophosphatase, sulfatase, xcex2-D-xylosidase, peptidase, aminopeptidase, trypsin, chymotrypsin, and phosphohydrolase. In a preferred embodiment, the bacterial enzyme is selected from the enterococcus specific enzymes consisting of xcex2-D-glucosidase, pyrrolidonyl arylamidase, and leucine aminopeptidase.
By xe2x80x9cnutrient indicatorxe2x80x9d is meant a molecule or substance containing a nutrient source attached to or conjugated with a moiety which produces either a detectable signal in a medium or an intermediate molecule which provides a detectable signal in the medium upon reacting with a developing agent. The two or more nutrient indicators are provided in an amount to support the growth of target bacteria. As target bacteria grow from the phase in which nutrients are accumulated for reproduction (lag phase) into the phase in which reproduction occurs at a relatively rapid rate (log phase), nutrition requirements change. Consequently, increasing amounts of nutrient indicators are metabolized and detectable signals or intermediates are produced. Nutrient sources may provide essential vitamins, minerals, trace elements, amino acid ingredients or carbon. The nutrient indicator may provide the primary carbon source to support substantial reproductive growth of target microbes until detectable characteristics are produced. Other nutrient sources may also be provided, so long as adequate selectivity and sensitivity of the medium is maintained. For examule, the nutrient indicator may be the primary source of carbon for the target bacteria. Alternatively, other carbon sources may be present (e.g. amino acids) which might be preferentially used by the target bacteria but the amount provided is such that not to reduce the specificity (and preferably, the sensitivity) of the medium.
The moiety attached to or conjugated with the nutrient source may be a detectable moiety or an intermediate molecule. A xe2x80x9cdetectable moietyxe2x80x9d is a molecule or substance which can either be covalently linked to a nutrient source or exist as a separate entity by itself. The detectable moiety does not cause or produce a detectable signal when it is covalently bonded to a nutrient source. However, when a bacterial enzyme hydrolyses the nutrient indicator, the detectable moiety is released and causes or produces a detectable signal. A detectable moiety may be a chromogen or a fluorogen. Fluorogens fluoresce upon exposure to an excitation light source. Fluorogens include, but are not limited to, 4-methylumbelliferone and 7-amido-4-methyl-coumarin moieties. Chromogens produce a color change observable in the visible range. Chromogens include, but are not limited to, o-nitrophenyl and bromo-chloro-indole moieties. O-nitrophenyl moieties produce a yellow color when released from the nutrient moiety. Bromo-chloro-indole moieties become blue when released from the nutrient moiety.
An xe2x80x9cintermediate moleculexe2x80x9d is a molecule or substance which can either be covalently linked to a nutrient source or exist as a separate entity by itself. Unlike a detectable moiety, an intermediate molecule, when released from the nutrient source, does not by itself immediately provide an easily detectable signal. It does provide a detectable signal, however, upon reacting with a developing agent. An exemplary intermediate molecule is xcex2-naphtylamide, which does not change the color of the medium when released from the nutrient indicator. However, the released xcex2-naphtylamide produces a pink to red color when mixed with a developing agent, p-dimethylaminocinnamaledhyde.
By xe2x80x9cdetectable signalxe2x80x9d is meant a characteristic change in a medium or sample that is observable or measurable by a physical, chemical, or biological means known to those skilled in the art. A detectable signal may be a change in emission or absorbance of visible or invisible light or radio waves at a certain wavelength, electrical conductivity, hybridization, enzymatic reaction, emission of gas, or odor. A detectable signal may also be a change in physical state such as between solid, liquid and gas. In preferred embodiments, detectable signals are changes in color or fluorescent emission of the medium.
Nutrient indicators for xcex2-glucosidase include, but are not limited to, resofuran-xcex2-D-glucopyranoside, p-nitrophenyl-xcex2-D-glucopyranoside, 5-bromo-4-chloro-3-indoxyl-xcex2-D-glucopyranoside, 6-bromo-2-naphtyl-xcex2-D-glucopyranoside, Rose-xcex2-D-glucopyranoside, VQM-Glc(2-{2-[4-(xcex2-D-glucopyranosyloxy)-3-methoxyl]vinyl)-1-methyl-quinolinium iodide, VBZTM-Gluc(2-{2-[4-(xcex2-D-glucopyranosyloxy)3-methoxylphenyl]vinyl}-3-methylbenzothiazolium iodide, and 4-methylumbelliferyl-xcex2-D-glucopyranoside.
Nutrient indicators for pyrrolidonyl arylamidase include, but are not limited to, L-pyroglutamic acid p-nitroanilide, L-pyroglutamic acid 7-amido-4-methyl-coumarin, and pyroglutamic acid xcex2-naphtylamide.
The invention also features a method of using the above described medium to sequentially detect the presence or absence of two or more bacterial enzymes in a test sample. The medium is inoculated with the test sample and incubated under conditions suitable for bacteria growth for a certain time period (preferably no more than 24 hours, more preferably no more than 18 hrs, even more preferably no more than 10 hours). The first detectable signal is used as an indication of the presence of the first bacterial enzyme in the test sample. The medium is then brought into contact with a developing agent and the second detectable signal is used as an indication of the presence of the second bacterial enzyme in the test sample.
By xe2x80x9cinoculatingxe2x80x9d is meant mixing the environmental, biological or clinical sample with a liquid medium or liquefied solid medium of this invention or bringing the sample into contact with a semi solid medium.
By xe2x80x9ctest samplexe2x80x9d is meant a piece, fraction, aliquot, droplet, portion, fragment, volume, or tidbit taken from a human or an animal test subject, or from soil, water, air or other environmental sources, or any other sources whose bacterial content needs to be determined. Clinical samples are taken from or coming from human sources. Examples include, but are not limited to, rectal and perirectal swabs, wound swabs, stool specimens, urine specimens, and blood. Environmental and biological samples are taken from or coming from a substance capable of supporting one or more life forms including yeast and bacteria. Examples include, but are not limited to, swabs taking from drinking water, food, utensils or equipment surfaces.
By xe2x80x9cbacteriaxe2x80x9d is mean one or more viable bacteria existing or co-existing collectively in a test sample. The term may refer to a single bacterium (e.g., Escherichia coli), a genus of bacteria (e.g. pseudomonads), a number of related species of bacteria (e.g. coliforms), an even larger group of bacteria having a common characteristic (e.g. all gram-negative bacteria), a group of bacteria commonly found in a food product, an animal, or human subject, or an environmental source, or a combination of two or more bacteria mentioned above. The bacteria include those described or referred to in Bergev""s Manual of Systematic Bacteriology, 1989, Williams and Wilkins, U.S.A., incorporated by reference herein.
The term xe2x80x9cEnterococcixe2x80x9d includes, but is not limited to, the following species of microorganisms: Enterococcus avium, E. casseliflavus, E. cerorum, E. columbae, E. dispar, E. durans, E. faecalis, E. faecium, E. gallinarum, E. hirae, E. malodoratus, E. mundtii, E. pseudoavium, E. raffinosus, E. saccharolyticus, E. seriolicida, E. solitarius, and E. sulfureus. This term is not meant to exclude species which have yet to be discovered but may later be identified and included in this genus by those skill in the art.
The term xe2x80x9cvancomycin-resistant Enterococcixe2x80x9d includes, but is not limited to, the following species of Enterococci which are able to grow in the presence or at least 16 xcexcg/ml vancomycin: Enterococcus casseliflavus, E. faecalis, E. faecium, and E. gallinarum. Among them, E. faecalis and E. faecium are the strains of clinical significance comprising more than 95% of clinical isolates. The term is not meant to exclude species which have yet to be discovered but may later be identified and included in this genus and shown to be resistant to vancomycin by those of skill in the art.
By xe2x80x9cselective agentsxe2x80x9d is meant antibiotics which prevent or inhibit the growth of fungi and gram negative bacteria and prevent microbes other than Enterococci from metabolizing the nutrient indicators. Selective agents include, but are not limited to, sodium azide, sodium chloride, thallium acetate, nalidixic acid, enoxacin, cinoxacin, ofloxacin, norfloxacin, gentamicin, neomycin, bile salts, lincomycin, colistin, ansiomycin, and cycloheximide. Preferably, it includes (per liter of medium) amikacin sulfate (e.g. about 0.005 to 0.015 mg), polymyxin B (about 0.002 to 0.015 mg), amphotericin B (about 0.001 to 0.030 mg), bacitracin (about 0.0004 to 0.0015 mg), cefotaxime (about 0.5 to 5 mg), and clindamycin (about 0.002 to 0.010 mg).
This invention features a medium for detecting the presence or absence of vancomycin-resistant Enterococci. Such a medium contains: (a) vancomycin in an amount sufficient to suppress the growth or vancomycin sensitive Enterococci; (b) one or more selective agents in an amount sufficient to suppress the growth of fungi, gram positive and gram negative bacteria other than Enterococci; c) a first nutrient indicator which provides a first detectable signal when cleaved by xcex2-glucosidase; and d) a second nutrient indicator which provides an intermediate molecule when cleaved by pyrrolidonyl arylamidase, wherein the intermediate molecule provides a second detectable signal upon reacting with a developing agent. Alternatively, the d) above is replaced by a second nutrient indicator which provides a second detectable signal when cleaved by pyrrolidonyl arylamidase, wherein the presence of both the first detectable signal and the second detectable signal is distinctively detectable from the presence of only one of the detectable signals.
In a preferred embodiment, the first nutrient indicator is selected from the group consisting of resofuran-xcex2-D-glucopyranoside, o-nitrophenyl-xcex2-D-glucopyranoside, p-nitrophenyl-xcex2-D-glucopyranoside, 5-bromo-4-chloro-3-indoxyl-xcex2-D-glucopyyranoside, 6-bromo-2-naphtyl-xcex2-D-glucopyranoside, Rose-xcex2-D-glucopyranoside, VQM-Glc(2-{2-[4-(xcex2-D-glucopyranosyloxy)-3-methoxyl]vinyl)-1-methyl-quinolinium iodide, VBZTM-Gluc(2-{2-[4-(xcex2-D-glucopyranosyloxy)-3-methoxylphenyl]vinyl}-3-methylbenzothiazolium iodide, and 4-metylumbelliferyl-xcex2-D-glucopyranoside.
In other preferred embodiments, the second nutrient indicator is pyroglutamic acid-xcex2-naphtylamide; the developing agent is p-dimethylaminocinnamaldehyde.
In a further preferred embodiment, the fist nutrient indicator is o-nitrohenyl-xcex2-D-glucopyranoside for enterococcus xcex2-glucosidase; the second nutrient indicator is pyroglutamic acid xcex2-naphtylamide for enterococcus pyrrolidonyl arylamidase; and the developing agent is p-dimethylaminocinnamaldehyde.
In another preferred embodiment, vancomycin is provided in an amount to inhibit the growth of both vancomycin sensitive Enterococci and non-Enterococci gram positive bacteria, e.g. 12 to 25 milligrams per liter or medium.
The invention also features a method of using the above described medium to detect the presence or absence of vancomycin-resistant Enterococci in a test sample. The medium is inoculated with the test sample and incubated under conditions suitable for Enterococci growth for a certain time period (preferably no more than 24 hours, more preferably no more than 18 hrs, even more preferably no more than 10 hours). After the detection of the first detectable signal (which indicates the presence of xcex2-glucosidase in the test sample), the medium is brought into contact with a developing agent and the second detectable signal is used as an indication for the presence of pyrrolidonyl arylamidase in the test sample. The presence of both the first and second detectable signals indicates that the sample contains vancomycin-resistant Enterococci. The testing medium does not have to be kept sterile, but, obviously must be free of viable target microbes, and the test procedure does not have to be performed in a sterile environment.
In a preferred embodiment, the medium is in powder form, which is liquified with sterile water or non-sterile water before a test sample is inoculated with the medium. The incubation may be performed at a variety of temperatures, but preferably carried out between 35xc2x0 C. and 45xc2x0 C.
The term xe2x80x9cliquifiedxe2x80x9d means substantially in liquid form, though it is also meant to include pulverized or homogenized samples of solid substances having at least a 10% liquid content. This phrase is meant to exclude a gelled medium, such as is found with agar.
In another preferred embodiment, the method uses an agar medium containing the first and second nutrient indicators. After the detection of the first detectable signal (which indicates the presence of xcex2-glucosidase in the test sample), a developing agent is added on top of the agar medium and the second detectable signal is used as an indication for the presence of pyrrolidonyl arylamidase in the test sample. The presence of both the first and second detectable signals indicates that the sample contains vancomycin-resistant Enterococci.
In yet another aspect, the invention features a method for quantifying the number of vancomycin-resistant Enterococci present in a sample by contacting the sample with the liquefied medium described above, placing the sample and medium mixture in containers, incubating the sample and medium mixture, observing the quantity and quality of detectable characteristic signals, and comparing the quantity of detectable characteristic signals with the most probable number (MPN) values. The MPN technique is based on probability statistics and the results from any type of an MPN analysis are directly related to the frequency of occurrence of a series of positive results that are most likely to occur when given numbers of organisms are present in a sample.
In preferred embodiments, the invention used the apparatus described by Croteau et al. in U.S. Ser. No. 08/557,529, hereby incorporated by reference.
Using the media and methods of this invention, a test sample containing only about 1-10 viable vancomycin-resistant Enterococci per ml can display detectable characteristic changes in 24 hour. The amount of oxygen and carbon dioxide in the medium, amount and type of enzyme inducer present, amount and type of selective agents present, amount of nutrients provided in the medium, and the relative health of the bacteria all affect the detection time. The addition of agents such as pyruvate, which may aid recovery of injured organisms, may increase the speed of detection. If large numbers of bacteria are present in the sample, more rapid detection is also possible. In this invention, the medium provided allows detection of 100-1000 cells/ml in less than 18 hours and 1-10 cells/ml of target microbes in less than 24 hours without cross reactivity from 107 cells of non-target microbes, at least 95% of the time.
This invention can be used in detection of vancomycin resistant Enterococci in such settings as hospitals, clinical and veternary laboratories, and nursing homes. Compared to the existing culture methods in detecting vancomycin resistant Enterococci in a sample, the method of this invention takes shorter time and does not require sterile medium preparation or multiple steps of culture isolation, biochemical identification, and antibiotic susceptibility confirmation. Furthermore, this invention does not require highly skilled medical technologists or microbiologists to perform the test. These advantages make this invention ideal for routine microbiological detection of vancomycin-resistant Enterococci.